6-羟基多巴胺诱导PC12细胞帕金森模型中GRP78表达的研究

The expression of glucose regulating protein 78 in a model of Parkinson's disease induced by 6-Hydroxydopamine in PC12 cells

目的探讨神经毒素6-羟基多巴胺(6-OHDA)诱导PC12细胞的帕金森(PD)模型中GRP78的表达。方法在建立6-OHDA诱导PC12细胞帕金森模型的基础上,MTT法测细胞存活率和Hoechst33342染色检测细胞凋亡。分别提取6-OHDA处理组和对照组细胞总蛋白,应用荧光差异凝胶电泳(Differentia Gel Electrophoresis,DIGE)技术获得蛋白点的差异表达信息,运用MALDI-TOF质谱鉴定出差异蛋白质。结果实验组细胞存活率为60%±4.8%,与对照组比较显著下降(P<0.05)。荧光染色可见细胞核呈固缩状或碎裂状的典型的凋亡形态学改变。DIGE分析发现6-OHDA组表达明显增高的一个蛋白质点,经质谱分析鉴定确认为GRP78。结论6-OHDA能够诱导PC12细胞凋亡,凋亡过程中GRP78表达增高,提示GRP78增高可能与PD的发病机制有关。

Objective To evaluate the expression level of glucose regulating protein 78（GRP78） in the model of Parkinson＇s disease（PD）induced by 6-Hydroxydopamine （6-OHDA） in PC12 cells. Methods After establishment of a model of PD induced by 6-OHDA in PC12 cells,MTT assay was applied to measure the cell vitality and Hoechst 33342 fluorescence staining was used to estimate the apoptotic percentage. Proteins of PC12 cells treated with and without 6-OHDA were extracted ,and then the maps of the proteins were established by 2D-DIGE（Differential Gel Electrophoresis）. The altered protein spots were identified with MALDI-TOF MS and database searching. Results The PC12 cells exposed to 6-OHDA showed a significant decrease in cell vitality（P〈0.05）. The nuclei of PC12 cells appeared condensed and fragmented which was consistent with nuclear morphology in Hoechst fluorescence staining. GRP78,a protein increased obviously after 6-OHDA treated,was identified by MALDI-TOF MS. Conclusion Our study indicated that 6-OHDA induced PC12 cells＇ apoptosis and the increasing expression of GRP78. It demonstrates that the increase of GRP78 be involved in the pathogenesis of PD.